Heparan sulfate (HS) is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation, and can facilitate viral infection (Esko and Selleck (2002) Annu. Rev. Biochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 22, 1-25). HS exerts its biological effects by interacting with the specific proteins involved in a given process (Capila and Lindhardt (2002) Angew. Chem. Int. Ed. 41, 390-412). HS is a highly charged polysaccharide consisting of 1→4-linked glucosamine and glucuronic/iduronic acid units that contain both N- and O-sulfo groups. Unique saccharide sequences within HS determine the specificity of the binding of HS to its target proteins (Linhardt (2003) J. Med. Chem. 46, 2551-2564). Heparin, a specialized form of HS, is a commonly used anticoagulant drug. Thus, new methods for the synthesis of heparin and HS attract considerable interest for those developing anticoagulant and other HS-related drugs having improved pharmacological effects.
Chemical synthesis has been the major route to obtain structurally defined heparin and HS oligosaccharides (Petitou and van Boeckel (2004) Angew. Chem. Int. Ed. 43, 3118-3133). One example of a chemically synthesized HS oligosaccharide is a synthetic pentasaccharide having antithrombin-binding properties marketed in the United States under the trade name ARIXTRA® (GlaxoSmithKline, Middlesex, United Kingdom). ARIXTRA® is a specific factor Xa inhibitor that is used clinically to prevent venous thromboembolic incidents during surgery.
Unfortunately, the total synthesis of heparin and HS oligosaccharides, larger than pentasaccharides, is difficult. HS analogues with 14 saccharide units inhibit the activity of thrombin, but these synthetic analogues are simplified hybrid molecules of HS oligosaccharides and highly sulfated glucose units (Petitou et al. (1999) Nature 398, 417-422; Dementiev et al. (2004) Nat. Struct. Biol. 11, 867-863) and are not the naturally occurring structures. Although the pursuit for the chemical synthesis of heparin and HS oligosaccharides (Avci et al. (2003) Curr. Pharm. Des. 9, 2323-2335) continues, it has become clear that chemical synthesis alone is currently incapable of generating most larger oligosaccharide structures. Thus, the application of HS biosynthetic enzymes for generating large heparin and HS oligosaccharides with desired biological activities offers a promising alternative approach.
Six classes of enzymes are involved in HS biosynthesis. HS is initially synthesized as a copolymer of D-glucuronic acid and N-acetylglucosamine (GlcNAc) through the action of D-glucuronyl and N-acetyl-D-glucosaminyltransferase (Lindahl et al. (1998) J. Biol. Chem. 273, 24979-24982). Next, a series of modifications take place, including N-deacetylation and N-sulfation (carried out by N-deacetylase/N-sulfotransferase) of the glucosamine residue to form N-sulfoglucosamine (GlcNS), C5 epimerization of glucuronic acid (carried out by epimerase) to form L-iduronic acid (IdoUA), 2-O-sulfation of IdoUA (carried out by 2-O-sulfotransferase (2-OST)), 6-O-sulfation of glucosamine (carried out by 6-O-sulfotransferase (6-OST)), and 3-O-sulfation of glucosamine (carried out by 3-O-sulfotransferase (3-OST)) (Sasisekharan et al. (2002) Nat. Rev. Cancer 2, 521-528). The reactions catalyzed by 2-OST, 6-OST, and 3-OST are shown in FIG. 1A.
Enzymes “in the pathway” for HS biosynthesis have been cloned and expressed, and have been employed in the synthesis of HS polysaccharides. Kuberan and Rosenberg (Balagurunathan et al. (2003) Nat. Biotechnol. 21, 1343-1346; Kuberan et al. (2003) J. Am. Chem. Soc. 125, 12424-12425; Balagurunathan et al. (2003) J. Biol. Chem. 278, 52613-52621) utilized these enzymes to synthesize an HS containing antithrombin binding sites with anticoagulant activity. Although this approach demonstrated for the first time the feasibility of enzymatic synthesis of HS, only about 1 μg of product was generated, making extensive structural characterization and biological studies impossible. Recently, Lindahl and colleagues reported an alternative chemoenzymatic approach for the synthesis of anticoagulant heparin from heparosan, the E. coli K5 capsular polysaccharide (Lindahl et al. (2005) J. Med. Chem. 48, 349-352). This method utilized the C5 epimerase to convert D-glucuronic acid to IdoUA, followed by the chemical persulfation and finally selective desulfation. Although this approach afforded approximately 5 g of a heparin-like polysaccharide with anticoagulant activity, unnatural saccharide units, such as 3-O-sulfo-D-glucuronic acid, were present in their product. This suggested a limitation in the selectivity of chemical sulfation/desulfation in HS synthesis. Further, the OST catalyzed sulfation reaction utilizes 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfur donor, producing adenosine 3′,5′-diphosphate (PAP). PAP can compete with PAPS for OST binding, which can result in inhibition of the sulfation reaction over time as PAP concentration increases in the reaction mixture.
Thus, developing an effective and highly selective approach for O-sulfation of polysaccharides remains an unmet need in the art for the large scale synthesis of HS.